Our broad aim is to define all of the signal transduction proteins that are part of the EpoR signaling complex and that are activated by Epo- stimulation, and to define their roles in controlling cell proliferation, erythroid differentiation, and prevention of apoptosis. An EpoR missing the C-terminal 100 amino acids supports Epo-dependent proliferation of Ba/F3 cells, yet several signal transduction proteins, particularly PI-3 kinase and SH-PTP1, are activated by more distal segments of the receptor. Presumably these and other signaling proteins are essential for erythroid differentiation and/or prevention of apoptosis. We showed that at least one protein-tyrosine kinase additional to JAK2 is involved in signaling by the EpoR, and we want to identify this kinase and determine its functions. We also showed that Y479 contains the principal binding site for PI-3 kinase and is essential for activation of MAP kinase, and we want to determine the function of these signaling proteins for differentiation of erythroid progenitors in vitro and in living mice. We showed that activation of the KIT receptor protein-tyrosine kinase induces tyrosine phosphorylation of the EpoR and that the EpoR is essential for KIT signaling in one cell line; thus, we want to determine how KIT signals through the EpoR. In several systems IGF-1 synergizes with Epo for erythroid differentiation, and we want to test whether the IGF-1 receptor, like KIT, signals through the EpoR. To accomplish these goals, we will generate a battery of EpoRs with specific mutations in their cytosolic domains; one focus will be on the extended Box 2 region essential for cell proliferation and KIT binding but not for JAK2 activation. In vitro and intact cell studies will determine sites necessary for binding and/or activation of specific signal transduction proteins. By expressing mutant receptors in a number of cell lines, in several types of primary erythroid progenitor cells, and, importantly in living mice in place of the normal EpoR, we can determine which EpoR segments are essential for preventing apoptosis, triggering cell proliferation, and inducing erythroid differentiation. Functions of these candidate proteins will be established by expressing or overexpressing wild-type or mutant proteins in cells expressing wild-type or mutant EpoRs. As part of these studies, we will generate mice in which one or both copies of the EpoR are replaced by mutants unable to bind and activate SH-PTP1 or PI-3 kinase.